Microorganism having acyltransferase activity and uses thereof

ABSTRACT

The present disclosure relates to a polypeptide having an acyltransferase activity or a microorganism including the same; a composition for preparing N-acetyl-L-methionine, the composition including the polypeptide or microorganism; and a method of preparing N-acetyl-L-methionine using the polypeptide or microorganism. Further, the present disclosure relates to a polynucleotide encoding the polypeptide and an expression vector including the polynucleotide. Since the microorganism including a novel acyltransferase according to the present disclosure has enhanced acyltransferase activity, this microorganism can be efficiently used for producing N-acetyl-L-methionine by acetylating L-methionine.

TECHNICAL FIELD

The present disclosure relates to a polypeptide having anacyltransferase activity or a microorganism including the same; acomposition for preparing N-acetyl-L-methionine, the compositionincluding the polypeptide or microorganism; and a method of preparingN-acetyl-L-methionine using the polypeptide or microorganism. Further,the present disclosure relates to a polynucleotide encoding thepolypeptide and an expression vector including the polynucleotide.

BACKGROUND ART

N-Acetylmethionine, which is a derivative of methionine, has similarefficacy to methionine, but it can reduce methionine-specific flavorsand can be added in a large amount compared to methionine when added tofoods. In the case of animals having a rumen, when methionine is used asa feed additive, it is first used by rumen microorganisms and thus isnot absorbed by the animals, whereas N-acetylmethionine is arumen-protected amino acid that is absorbed after passing through therumen and reaching the intestine. The degradation resistance ofN-acetyl-DL-methionine at the rumen is known (Amos et al., Journal ofAnimal Science, 1974, 39(3), pp. 612-617). Accordingly, the productionof N-acetylmethionine has industrial value, and particularly, it ispreferable to provide an L-type amino acid having a high absorption rateand high bioavailability when used as a feed additive, and thereforeresearch and development of N-acetyl-L-methionine is required. In thecase of enzymes reported to convert methionine to N-acetylmethionine,YncA of Escherichia coli is unique (U.S. Pat. No. 8,143,031 B2). In thisdocument, it was found that acetyl-CoA was used indirectly through aDTNB analysis method to measure the inactivity of YncA, and it was notfound that N-acetylmethionine was produced. Further, it has not beenfound that N-acetylmethionine is actually produced by the transformanttransformed by YncA.

DISCLOSURE Technical Problem

The present inventors have conducted continuous efforts to produceN-acetyl-L-methionine through a microbial fermentation or an enzymaticreaction, and thus have searched for microorganisms capable of producingN-acetyl-L-methionine and discovered six new acyltransferases. Further,they have found that N-acetyl-L-methionine can be produced economicallyat a high concentration using the novel acyltransferase of the presentdisclosure or a microorganism expressing the novel acyltransferase, ascompared with a known acyltransferase. Based on this finding, thepresent disclosure has been completed.

Technical Solution

An object of the present disclosure is to provide a microorganism havingan acyltransferase activity, the microorganism including a polypeptiderepresented by an amino acid sequence of any one of SEQ ID NOS. 1 to 6or an amino acid sequence having 90% or more homology to the amino acidsequence.

Another object of the present disclosure is to provide a polypeptidehaving an acetyltransferase activity, the polypeptide being representedby an amino acid sequence of any one of SEQ ID NOS. 1 to 6 or an aminoacid sequence having 90% or more homology to the amino acid sequence.

Still another object of the present disclosure is to provide apolynucleotide encoding the polypeptide.

Still another object of the present disclosure is to provide anexpression vector including the polynucleotide.

Still another object of the present disclosure is to provide acomposition for preparing N-acetyl-L-methionine from L-methionine, thecomposition including, as an active ingredient: (i) the microorganism ora culture thereof; (ii) the polypeptide; or a combination thereof.

Still another object of the present disclosure is to provide a method ofpreparing N-acetyl-L-methionine, including: acetylating L-methionineusing (i) the microorganism or a culture thereof; (ii) the polypeptide;or a combination thereof.

Advantageous Effects

Since the microorganism including a novel acyltransferase according tothe present disclosure has enhanced acyltransferase activity, thismicroorganism can be efficiently used for producingN-acetyl-L-methionine by acetylating L-methionine.

BEST MODE FOR INVENTION

In order to accomplish the above objects, an aspect of the presentdisclosure provides a microorganism having acyltransferase activity, themicroorganism including a polypeptide represented by an amino acidsequence of any one of SEQ ID NOS. 1 to 6 or an amino acid sequencehaving 90% or more homology thereto.

The term “acyltransferase” used in the present disclosure refers to anenzyme having an activity of transferring an acyl group from a donor toa receptor. In the present disclosure, the donor is not limited as longas it can provide an acyl group to a receptor, but may specifically beacetyl coenzyme A (acetyl-CoA). Further, as used herein, the receptor isnot limited as long as it can receive an acyl group from a donor, butmay specifically be L-methionine.

Specifically, the acyltransferase may be derived from genus Pseudomonas,genus Bacillus, genus Enterobacter, genus Pseudovibrio, genus Yarrowia,or genus Corynebacterium. More specifically, the acyltransferase may bederived from Pseudomonas putida, Bacillus subtilis, Enterobacter sp.638, Pseudovibrio sp. FO-BEG1, Yarrowia lipolytica, or Corynebacteriumglutamicum.

Specifically, the acyltransferase may be a polypeptide having an aminoacid sequence of any one of SEQ ID NOS. 1 to 6. Further, theacyltransferase may be a polypeptide having an amino acid sequencehaving 70% or more, 80% or more, or 90% or more homology to an aminoacid sequence of any one of SEQ ID NOS. 1 to 6 and having anacyltransferase activity substantially the same as or corresponding tothat of the amino acid sequence of any one of SEQ ID NOS. 1 to 6.Further, the amino acid sequence having such homology and having anacyltransferase activity substantially the same as or corresponding tothat of the amino acid sequence of any one of SEQ ID NOS. 1 to 6 may bean amino acid sequence, a part of which is deleted, transformed,substituted, or added. It is obvious that this case may also be includedin the scope of the present disclosure.

The term “homology” used in the present disclosure refers to the degreeof identity of base or amino acid residues between sequences afteraligning both amino acid sequences or base sequences of a gene encodinga polypeptide in a specific comparison region to be matched with eachother as much as possible. When the homology is sufficiently high,expression products of the corresponding gene may have the same orsimilar activity. The percentage of the sequence identity can bedetermined using a known sequence comparison program, for example, BLAST(NCBI), CLC Main Workbench (CLC bio), MegAlign (DNA STAR Inc), or thelike.

The term “microorganism having an acyltransferase activity” used in thepresent disclosure refers to a microorganism producing anacyltransferase in vivo or in vitro. Specifically, since themicroorganism of the present disclosure includes a polypeptide having anamino acid sequence of any one of SEQ ID NOS. 1 to 6 and thus has anacyltransferase activity, it can transfer an acyl group to a receptor.More specifically, the microorganism of the present disclosure hasacetyltransferase activity to L-methionine and thus can produceN-acetyl-L-methionine. In the present disclosure, N-acetyl-L-methionineand N-acetylmethionine are used interchangeably.

Additionally, the microorganism of the present disclosure includes notonly microorganisms which inherently contain a polypeptide having anamino acid sequence of any one of SEQ ID NOS. 1 to 6, but alsomicroorganisms in which the activity of the polypeptide is enhanced ascompared with an intrinsic activity. That is, the production capacity ofthe acyltransferase can be imparted or enhanced by natural oranthropogenic mutagenesis or species modification. The term“enhancement” of polypeptide activity, as used herein, refers toimproving the active state of the polypeptide included in themicroorganism. Enhancement of polypeptide activity is not limited aslong as it can enhance the activity of each polypeptide, such as theenhancement of the activity of the target polypeptide, as compared witha natural state or to a non-variation state of the polypeptide. Forexample, the enhancement of polypeptide activity may be performed by amethod selected from i) increasing the number of copies of apolynucleotide encoding each polypeptide, ii) modifying an expressionsequence to increase the expression of the polynucleotide, iii)modifying the polynucleotide sequence on the chromosome to enhance theactivity of each polypeptide, and iv) a combination thereof.Specifically, the enhancement of polypeptide activity may be performedby a method selected from a method of inserting a polynucleotideincluding a nucleotide sequence encoding each polypeptide into achromosome, a method of introducing the polynucleotide into amicroorganism through a vector system, a method of introducing apromoter exhibiting an improved activity upstream of a base sequenceencoding each polypeptide or introducing each of the mutatedpolypeptides into a promoter, a method of modifying the nucleotidesequence in the 5′-UTR region, and a method of introducing a mutant ofthe base sequence encoding each polypeptide, but the present disclosureis not limited thereto.

Further, in the present disclosure, the microorganism having anacyltransferase activity may be used regardless of the origin of themicroorganism as long as it has an acyltransferase activity. Forexample, the microorganism may be Escherichia sp., Corynebacterium sp.,Saccharomyces sp., or Yarrowia sp. More specifically, the microorganismmay be Escherichia coli, Corynebacterium glutamicum, Saccharomycescerevisiae, or Yarrowia lipolytica, but is not limited thereto.

Further, in the present disclosure, the microorganism having anacyltransferase activity may be a microorganism having enhanced cellmembrane permeability of a donor and/or a receptor. As the method ofincreasing the cell membrane permeability, a known method may be used.Specifically, processes of freezing and thawing the microorganism may berepeated, but the present disclosure is not limited thereto.

Further, in the present disclosure, the microorganism having anacyltransferase activity may biosynthesize a receptor to which an acylgroup is transferred from a donor, but the present disclosure is notlimited thereto. Specifically, the microorganism of the presentdisclosure may produce N-acetyl-L-methionine even when it is cultured ina medium to which L-methionine is not added because it has an ability ofproducing L-methionine that is an acceptor of an acyl group.

Further, in the present disclosure, the microorganism having anacyltransferase activity may be a mutant microorganism into which aknown mutation is introduced with respect to a related mechanism such asa biosynthesis-related pathway or a substrate releasing capacity-relatedmechanism in order to enhance N-acetyl-L-methionine production abilityseparately from the acyltransferase.

An aspect of the present disclosure provides a polypeptide having anacetyltransferase activity, the polypeptide being represented by anamino acid sequence of any one of SEQ ID NOS. 1 to 6 or an amino acidsequence having 90% or more homology to the amino acid sequence.Specifically, the acetyltransferase activity may be an acetyltransferaseactivity to L-methionine.

The polypeptide is as described above.

Another aspect of the present disclosure provides a polynucleotideencoding the polypeptide having an acetyltransferase activity, thepolypeptide being represented by an amino acid sequence of any one ofSEQ ID NOS. 1 to 6 or an amino acid sequence having 90% or more homologyto the amino acid sequence. The polypeptide is as described above.

For example, the polynucleotide may be a base sequence of any one of SEQID NOS. 7 to 12, but is not limited thereto. Further, the polynucleotidemay include a base sequence encoding the same amino acid sequence due togenetic code degeneracy, and a mutant thereof. For example, thepolynucleotide may be modified to have an optimal codon depending on themicroorganism used.

Specifically, the polynucleotide may be a base sequence encoding anamino acid sequence having 70% or more, 80% or more, or 90% or morehomology to the above base sequence and having an acyltransferaseactivity substantially the same as or corresponding to that of the abovebase sequence. Further, the polynucleotide may be a probe that can beprepared from a known gene sequence, for example, it may be a sequenceencoding a polypeptide having an acyltransferase activity byhybridization with a complementary sequence to all or part of the basesequence under stringent conditions. Here, the “stringent conditions”refer to conditions where a specific hybrid is formed and a non-specifichybrid is not formed. For example, the stringent conditions may includea condition where genes having high homology, for example, genes having80% or more, specifically 90% or more, more specifically 95% or more,further specifically 97% or more, or particularly specifically 99% ormore homology are hybridized and genes having lower homology are nothybridized, and a condition where cleaning is performed one time,specifically two or three times under a salt concentration andtemperature corresponding to 60° C., 1×SSC, 0.1% SDS, specifically 60°C., 0.1×SSC, 0.1% SDS, and more specifically 68° C., 0.1×SSC, 0.1% SDS,which are cleaning conditions for conventional hybridization, (Sambrooket al., Molecular Cloning: A Laboratory Manual, 3rd Ed., Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y. (2001)).

The probe used in the hybridization may be a part of a complementarysequence of the base sequence. This probe may be prepared by PCR using agene fragment including such a base sequence as a template by using anoligonucleotide prepared based on a known sequence as a primer. Forexample, as the probe, a gene fragment having a length of about 300 bpmay be used. More specifically, when a gene fragment having a length ofabout 300 bp is used as the probe, as cleaning conditions forhybridization, 50° C., 2×SSC, and 0.1% SDS are exemplified.

Genes, polypeptide sequences encoded by the genes, and promotersequences, which are used in the present disclosure, may be obtainedfrom known databases, for example, GenBank of NCBI. However, the presentdisclosure is not limited thereto.

Still another aspect of the present disclosure provides an expressionvector including the polynucleotide.

The expression vector including the polynucleotide according to thepresent disclosure is an expression vector capable of expressing atarget protein in a suitable host cell, and refers to a polynucleotideproduct including an essential control element operably linked so as toexpress a polynucleotide insert. Target proteins may be obtained bytransforming or transfecting the prepared recombination vector in thehost cell.

The expression vector including the polynucleotide according to thepresent disclosure is not particularly limited, but includes Escherichiacoli-derived plasmids (pYG601BR322, pBR325, pUC118, and pUC119),Bacillus subtilis-derived plasmids (pUB110 and pTP5), yeast-derivedplasmids (YEp13, YEp24, and YCp50), and Ti-plasmids that can be used foragrobacterium-mediated transformation. Specific examples of phage DNAinclude λ-phages (Charon4A, Charon21A, EMBL3, EMBL4, lambda gt10, lambdagt11, and lambda ZAP). Further, virus vectors derived from animalviruses such as retrovirus, adenovirus, and vaccinia virus, insectviruses such as baculovirus, double-stranded plant virus (for example,CaMV), single-stranded virus, or Geminiviruses may also be used.

Moreover, as the vector of the present disclosure, a fusion plasmid (forexample, pJG4-5) to which nucleic acid expression activating protein(for example, B42) is linked may be used. Further, in order tofacilitate the purification of a target protein recovered in the presentdisclosure, the plasmid vector may further include other sequences asnecessary. Such a fusion plasmid may include GST, GFP, His-tag, orMyc-tag as a tag, but the fusion plasmid of the present disclosure isnot limited to the above examples.

Further, in the production of the fusion protein, a chromatographyprocess may be used, and in particular, the fusion protein may bepurified by affinity chromatography. For example, whenglutathione-S-transferase is fused, glutathione, which is a substrate ofthis enzyme, may be used. When hexahistidine is used, a desired targetprotein can be easily recovered using a Ni-NTA His-conjugated resincolumn (Novagen, USA).

In order to insert the polynucleotide of the present disclosure as avector, a method of cleaving purified DNA with a suitable restrictionenzyme and inserting the cleaved DNA into a restriction site or acloning site of an appropriate vector DNA may be used.

The polynucleotide encoding the polypeptide of the present disclosuremay be operably linked to a vector. The vector of the present disclosuremay additionally include a cis-element such as an enhancer, a splicingsignal, a poly A addition signal, a selection marker, and a ribosomebinding sequence (SD sequence), in addition to the promoter and nucleicacid of the present disclosure. Examples of the selection marker mayinclude chloramphenicol resistance, ampicillin resistance, dihydrofolatereductase, and neomycin resistance, but additional components operablylinked by the above examples are not limited. The term “transformation”as used herein refers to a phenomenon of introducing DNA into the hostcell to allow the DNA to serve as a factor of a chromosome or to bereplicated by chromosome integration completion and introducing externalDNA into a cell to cause an artificial genetic change.

An expression vector including a polynucleotide encoding the polypeptideof the present disclosure or a part of the expression vector may beintroduced into a host cell. Here, a part of the expression vectorrefers to a portion of the expression vector, the portion including aportion of the polynucleotide encoding the polypeptide of the presentdisclosure so as to impart the acyltransferase activity into the hostcell. For example, T-DNA of Ti-plasmid transferred into the host cell inagrobacterium-mediated transformation may be exemplified, but thepresent disclosure is not limited thereto.

Any transformation method may be used for the transformation method ofthe present disclosure, and may be easily carried out according to amethod known in the art. Generally, the transformation method mayinclude a CaCl₂ precipitation method, a Hanahan method in which dimethylsulfoxide (DMSO) is used as a reduction material in the CaCl₂precipitation method to increase efficiency, an electroporation method,a CaPO₄ precipitation method, a protoplasm fusion method, a stirringmethod using silicon carbide fiber, an agrobacterium-mediatedtransformation method, a transformation method using PEG, a dextransulfate-mediated transformation method, a lipofectamine-mediatedtransformation method, and a drying/inhibition-mediated transformationmethod. The method of transforming a vector including a polynucleotideencoding the polypeptide of the present disclosure is not limited to theabove examples, and transformation or transfection methods commonly usedin the art may be used without limitation.

The kind of host cells used in the preparation of a transformant in thepresent disclosure is not particularly limited as long as thepolynucleotide of the present disclosure is expressed. Specific examplesof the host cells used in the present disclosure may include bacteria ofgenus Escherichia such as E. coli; bacteria of genus Bacillus such asBacillus subtilis; bacteria of genus Pseudomonas such as Pseudomonasputida; yeasts such as Saccharomyces cerevisiae and Schizosaccharomycespombe; animal cells; plant cells; and insect cells. Specific examples ofEscherichia coli strains that can be used in the present disclosure mayinclude CL41(DE3), BL21, and HB101, and specific examples of Bacillussubtilis strains that can be used in the present disclosure may includeWB700 and LKS87.

The transformant into which an expression vector including thepolynucleotide of the present disclosure is introduced may be in theform of a transformed cell or an organism.

As the promoter in the present disclosure, any promoter may be used aslong as it is capable of expressing the polynucleotide of the presentdisclosure in the host cell. For example, Escherichia coli orphage-derived promoters such as trp promoter, lac promoter, PL promoter,and PR promoter; Escherichia coli-infected or phage-derived promoterssuch as T7 promoter; CaMV35S, MAS or histone promoter; and cj7 promoter(Korean Patent Application Publication No. 10-2004-0107215) may be used.Further, artificially modified promoters such as tac promoter may alsobe used.

Still another aspect of the present disclosure provides a method ofpreparing N-acetyl-L-methionine, including: acetylating L-methionineusing (i) the microorganism of the present disclosure or a culturethereof; (ii) the polypeptide of the present disclosure; or acombination thereof.

Specifically, the method includes: preparing N-acetyl-L-methionine,including: acetylating L-methionine using (i) the microorganism of thepresent disclosure or a culture thereof; (ii) the polypeptide of thepresent disclosure; or a combination thereof; and recoveringN-acetyl-L-methionine, which is the acetylated L-methionine.

In the present disclosure, the “culturing” refers to growing themicroorganism under suitably controlled environmental conditions.

The culturing process of the present disclosure may be performedaccording to a suitable medium and culture conditions, which are knownin the art. Such a culturing process may be easily adjusted by thoseskilled in the art depending on the strain to be selected. In the abovemethod, the process of culturing the microorganism is not particularlylimited, but may be conducted by a batch culture method, a continuousculture method, or a fed-batch culture method, which is known in theart. The medium used for culturing the microorganism of the presentdisclosure and other culture conditions may be used without anyparticular limitation as long as it can be used for culturing generalmicroorganisms. Specifically, the microorganism of the presentdisclosure may be cultured in a general medium including a carbonsource, a nitrogen source, a phosphorus source, an inorganic compound,amino acid, and/or vitamin while controlling temperature, pH, and thelike under aerobic conditions.

In the present disclosure, examples of the carbon sources may include,but are not limited to, carbohydrates such as glucose, fructose,sucrose, maltose, mannitol, and sorbitol; alcohols such as saccharidealcohol, glycerol, pyruvic acid, lactic acid, and citric acid; organicacids; and amino acids such as glutamic acid, methionine, and lysine.Further, natural nutrient sources such as starch hydrolysate, molasses,blackstrap molasses, rice bran, cassava, sugar cane residues, and cornimmersion liquids may be used. Specifically, carbohydrates such asglucose and sterilized pretreated molasses (that is, molasses convertedto reducing sugars) may be used, and suitable amounts of other carbonsources may be variously used without limitation. These carbon sourcesmay be used alone or in a combination of two or more.

Examples of the nitrogen sources may include inorganic nitrogen sourcessuch as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate,ammonium phosphate, ammonium carbonate, and ammonium nitrate; andorganic nitrogen sources, such as amino acids such as glutamic acid,methionine and glutamine, peptone, NZ-amine, meat extracts, yeastextracts, malt extracts, corn immersion liquids, casein hydrolysates,fish or degradation products thereof, and defatted soybean cake ordegradation products thereof. These nitrogen sources may be used aloneor in a combination of two or more. However, the present disclosure isnot limited thereto.

Examples of the phosphorus sources may include potassium phosphate,potassium phosphite, and sodium-containing salts thereof. Examples ofthe inorganic compound may include sodium chloride, calcium chloride,iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, andcalcium carbonate, and may further include, but are not limited thereto,amino acids, vitamins, and/or suitable precursors. These media orprecursors may be added to a culture in a batch or continuous manner,but are not limited thereto.

In the present disclosure, the pH of a culture may be adjusted by addinga compound such as ammonium hydroxide, potassium hydroxide, ammonia,phosphoric acid, or sulfuric acid to a culture in an appropriate mannerduring the culturing of microorganisms. Further, during the culturing ofmicroorganisms, the formation of bubbles may be suppressed by using adefoaming agent such as aliphatic polyglycol ester. Further, oxygen oroxygen-containing gas may be injected into a culture in order tomaintain the aerobic state of the culture, or nitrogen, hydrogen, orcarbon dioxide gas may be injected into the culture in order to maintainthe anaerobic and non-aerobic states of the culture without injectingthe gas.

The temperature of the culture varies depending on the microorganism ofthe present disclosure. Specifically, the temperature thereof may be 20°C. to 50° C., more specifically, 25° C. to 40° C., but is not limitedthereto. The culturing period may be continued until the amount of adesired useful material is obtained. Specifically, the culturing periodmay be 10 hours to 100 hours, but is not limited thereto.

Further, the term “culture” as used herein refers to a product obtainedafter culturing the microorganism of the present disclosure. The cultureincludes both a form containing microorganisms and a form in whichmicroorganisms are removed by centrifugation or the like in a culturesolution containing the microorganisms.

Further, in the present disclosure, L-methionine may be produced by themicroorganism of the present disclosure or may be added to the medium.

Further, in the step of recovering N-acetyl-L-methionine in the presentdisclosure, targeted N-acetyl-L-methionine may be recovered from theculture solution using a suitable method known in the related artaccording to the method of culturing the microorganism of the presentdisclosure, for example, a batch culture method, a continuous culturemethod, or a fed-batch culture method. For example, centrifugation,filtration, anion exchange chromatography, crystallization, HPLC, andthe like may be used, and a combination of suitable methods known in theart may be used.

The recovering step may include a separating step and/or a purifyingstep.

Still another aspect of the present disclosure provides a compositionfor preparing N-acetyl-L-methionine from L-methionine, the compositionincluding, as an active ingredient: (i) the microorganism of the presentdisclosure or a culture thereof; (ii) the polypeptide of the presentdisclosure; or a combination thereof. The microorganism, culture,polypeptide, and N-acetyl-L-methionine are as described above.

MODE FOR INVENTION

Hereinafter, the present disclosure will be described in more detailwith reference to Examples. However, these Examples are onlyillustrative of the present disclosure, and the scope of the presentdisclosure is not limited to these Examples.

Example 1: Selection of Novel Enzyme for Producing N-acetylmethionineExample 1-1: Selection of Microorganism for ProducingN-acetyl-L-methionine

As microorganisms having an ability of producing N-acetyl-L-methionine,N-acetylmethionine spots, which are products, were confirmed from sixkinds of randomly selected microorganism culture solutions (Pseudomonasputida, Bacillus subtilis, Enterobacter sp. 638, Pseudovibrio sp.FO-BEG1, Yarrowia lipolytica PO1f (ATCC MYA-2613™), and Corynebacteriumglutamicum ATCC 13032).

Specifically, Pseudomonas putida, Bacillus subtilis, Enterobacter sp.638, or Yarrowia lipolytica PO1f was inoculated in a 14 mL disposableculture tube containing 3 mL of a liquid YPD medium (1% yeast extract,2% bacto-peptone, 2% glucose), and was shake-cultured for 24 hours underconditions of 30° C. and 200 rpm to obtain a seed culture solution. 1 mLof the seed culture solution was inoculated in a 250 mL corner-baffleflask filled with 24 mL of the liquid YPD medium containing 0.5% (5 g/L)of methionine, and was shake-cultured for 24 hours under conditions of30° C. and 200 rpm.

Further, Pseudovibrio sp. FO-BEG1 was inoculated in a 14 mL disposableculture tube containing 3 mL of a liquid BACTO Marine broth (Difco 2216)medium, and was shake-cultured for 24 hours under conditions of 28° C.and 200 rpm to obtain a seed culture solution. 1 mL of the seed culturesolution was inoculated in a 250 mL corner-baffle flask filled with 24mL of the liquid BACTO Marine broth (Difco 2216) medium containing 0.5%(5 g/L) of methionine, and was shake-cultured for 24 hours underconditions of 28° C. and 200 rpm.

Further, Corynebacterium glutamicum ATCC 13032 was inoculated in a 14 mLdisposable culture tube containing 3 mL of a dedicated composite liquidmedium (ingredients shown below), and was shake-cultured for 24 hoursunder conditions of 30° C. and 200 rpm to obtain a seed culturesolution. 1 mL of the seed culture solution was inoculated in a 250 mLcorner-baffle flask filled with 24 mL of the dedicated composite liquidmedium containing 0.5% (5 g/L) of methionine, and was shake-cultured for24 hours under conditions of 37° C. and 200 rpm.

<Composite Liquid Medium>

20 g glucose, 10 g peptone, 5 g yeast extract, 1.5 g urea, 4 g KH₂PO₄, 8g K₂HPO₄, 0.5 g MgSO₄7H₂O, 100 μg biotin, 1000 μg thiamine HCl, 2000 μgcalcium pantothenate, 2000 μg nicotinamide, and 25 mg kanamycin (basedon 1 L distilled water)

After completion of the culturing, the culture solution was left in anoven at 50° C. overnight to be concentrated, and 1 μL of theconcentrated culture solution was analyzed using thin layerchromatography. 10 mM of a N-acetyl-L-methionine reagent (Sigma Aldrich01310) was used as a control group. The same spot as the control groupwas confirmed from the culture liquid concentrate of the above six kindsof microorganisms.

From the above results, it was predicted that each of the six kinds ofmicroorganisms having an ability of producing N-acetyl-L-methioninecontains an acyltransferase including L-methionine as a substrate.

Example 1-2: Confirmation of Novel Acyltransferase Sequence

Methionine acyltransferase was selected from the six kinds ofmicroorganisms selected in Example 1-1.

Heterogeneous homology searches for the selected proteins were performedby restricting the database to species of the microorganism based on anamino acid sequence with YncA known as a conventional methionineacyltransferase. As a result, all of the six kinds of searched enzymeswere found to have low homology with the YncA amino acid sequence, andthe homology of the Enterobacter sp. 638-derived acyltransferasesequence, which had the highest homology, was 82%. Homologies of therespective yeasts with YncA are given in Table 1.

From the above results, it is inferred that the above kinds ofpolypeptides are polypeptides having novel acyltransferase activitylower than the homology of a conventional acyltransferase, and themicroorganisms including these polypeptides are also microorganismshaving novel acyltransferase activity which has not been previouslyknown.

TABLE 1 Homology (%) with YncA amino acid sequence Pseudomonasputida-derived 65 acyltransferase Bacillus subtilis-derivedacyltransferase 28 Enterobacter sp. 638-derived 82 acyltransferasePseudovibrio sp. FO-BEG1-derived 46 acyltransferase Yarrowia lipolyticaPO1f-derived 29 acyltransferase Corynebacterium glutamicum-derived 28acyltransferase

Example 2: Production of N-Acetylmethionine Through In Vitro EnzymeConversion Using Novel Acyltransferase Example 24: Preparation of NovelAcyltransferase-Introduced Escherichia coli

Pseudomonas-derived, Bacillus-derived, Enterobacter-derived, andPseudovibrio-derived novel acyltransferase genes (SEQ ID NOS. 7, 8, 9,and 10) were synthesized as codons optimized for Escherichia coli basedon amino acid sequences (SEQ ID NOS. 1, 2, 3 and 4) of the respectiveenzymes.

In order to obtain a Yarrowia-derived novel acyltransferase gene, gDNAof Yarrowia lipolytica PO1f (ATCC MYA-2613™) was extracted. A gene (SEQID NO. 11) of a desired size and an amino acid sequence (SEQ ID NO. 5)of a desired size were obtained by performing a polymerase chainreaction using primers 1 and 2 with the gDNA as a template.

In order to obtain a Corynebacterium-derived novel acyltransferase gene,Corynebacterium glutamicum ATCC 13032 was smeared on a solid medium withstreaks and then cultured overnight to obtain colonies. A gene (SEQ IDNO. 12) having a size of about 0.5 kb and an amino acid sequence (SEQ IDNO. 6) having a size of about 0.5 kb were obtained by performing PCR(polymerase chain reaction) using primers 3 and 4 with the one colony asa template. In this case, the PCR was performed by conductingdenaturation at 94° C. for 5 minutes, repeating denaturation at 94° C.for 30 seconds, annealing at 56° C. for 30 seconds and polymerization at72° C. for 1 minute 30 times, and then conducting a polymerizationreaction at 72° C. for 7 minutes.

The six kinds of DNAs were treated with restriction enzymes NdeI andXbaI and then ligated to a pUCtk vector treated with the samerestriction enzymes. The prepared recombinant plasmid was transformed byapplying thermal shock to Escherichia coli DH5a at 42° C. for 90seconds, and then those was smeared in an LB solid medium containingkanamycin and cultured overnight at 37° C. One colony obtained in theculture was inoculated in 3 mL of an LB liquid medium containingkanamycin and cultured overnight, and then the recombinant plasmid wasrecovered using a plasmid miniprep kit (Bioneer, Korea). Sequenceinformation of the recovered recombinant plasmid was confirmed bysequencing (Macrogen, Korea), and the plasmids were named pUCtk-ppmat,pUCtk-bsmat, pUCtk-entmat, pUCtk-pvmat, pUCtk-ylmat, and pUCtk-cgmat.

The colonies surviving after introducing the recombinant plasmid intoEscherichia coli BL21 (DE3) using thermal shock and smearing this in anLB solid medium containing kanamycin were selected as transformants. Theselected transformants were named BL21(DE3)/pUCtk-ppmat (or E. coliCC03-9001), BL21(DE3)/pUCtk-bsmat (or E. coli CC03-9002),BL21(DE3)/pUCtk-entmat (or E. coli CC03-9003), BL21(DE3)/pUCtk-pvmat (orE. coli CC03-9004), and BL21(DE3)/pUCtk-ylmat (or E. coli CC03-9005),BL21(DE3)/pUCtk-cgmat (or E. coli CC03-9006). Further, the transformantswere deposited to the Korean Culture Center of Microorganisms (KCCM) onJul. 15, 2016 under the Budapest Treaty, and received the depositnumbers KCCM11863P, KCCM11864P, KCCM11865P, KCCM11866P, KCCM11867P, andKCCM11868P, respectively, according to the above-described order.

In order to examine the N-acetylmethionine producing capacity of theselected transformed Escherichia co/i, one colony was inoculated in 3 mLof an LB liquid medium containing 25 mg/L of kanamycin and 0.2% (w/v) ofglucose, cultured at 37° C. for 5 hours, and then further cultured for 3hours with the addition of methionine up to 2% (w/v), so as to obtain 1μL of a culture solution. The production of N-acetylmethionine waspreviously examined by thin layer chromatography (TLC) analysis using 1μL of the culture solution. Spots presumed to be N-acetylmethionine werefound in the entire culture solution, the concentration of the spotsincreased in the order of BL21(DE3)/pUCtk-ppmat, BL21(DE3)/pUCtk-entmat,BL21(DE3)/pUCtk-bsmat, BL21(DE3)/pUCtk-cgmat, BL21(DE3)/pUCtk-pvmat, andBL21(DE3)/pUCtk-ylmat.

TABLE 2 Primer SEQ ID No. Sequence NO. 1 5′-ATCCATATGAAGATATCTCCAGAACCC13 2 5′-TACTCTAGACTAGTCACTCCTCGTGTC 14 3 5′-ATCCATATGGTTGAAAGAGACTTCAC15 4 5′-TACTCTAGATTAGGAACTTTGAGCTTG 16 5 5′-ATCatgagccatgaaatt 17 65′-GCACTGCAGtcacgccggttccgc 18 7 5′-ATCatgaccctgcgcctg 19 85′-GCACTGCAG tcagctcagttcgcg 20 9 5′-ATCATGATCATCCGCCAT 21 105′-GCACTGCAGTCAGATAGCGTCCGG 22 11 5′-ATCatgaaactgcgtcag 23 125′-GCACTGCAGttattcatgcgcgag 24 13 5′-ATCATGAAGATATCTCCA 25 145′-GCACTGCAGCTAGTCACTCCTCGT 26 15 5′-ATCATGGTTGAAAGAGAC 27 165′-GCACTGCAGTTAGGAACTTTGAGC 28 17 5′-tgcactagtatgagccatgaaatt 29 185′-tgcctcgagtcacgccggttccgc 30 19 5′-tgcactagtatgaccctgcgcctg 31 205′-tgcctcgagtcagctcagttcgcg 32 21 5′-tgcactagtATGATCATCCGCCAT 33 225′-GCACTGCAGTCAGATAGCGTCCGG 34 23 5′-tgcactagtatgaaactgcgtcag 35 245′-tgcctcgagttattcatgcgcgag 36 25 5′-tgcactagtATGAAGATATCTCCA 37 265′-tgcctcgagCTAGTCACTCCTCGT 38 27 5′-tgcactagtATGGTTGAAAGAGAC 39 285′-tgcctcgagTTAGGAACTTTGAGC 40 29 5′-tgcggtaccatgagccatgaaatt 41 305′-ctaggtacctcacgccggttccgc 42 31 5′-tgcggtaccatgaccctgcgcctg 43 325′-ctaggtacctcagctcagttcgcg 44 33 5′-tgcggtaccATGATCATCCGCCAT 45 345′-ctaggtaccTCAGATAGCGTCCGG 46 35 5′-tgcggtaccatgaaactgcgtcag 47 365′-ctaggtaccttattcatgcgcgag 48 37 5′-tgcggtaccATGAAGATATCTCCA 49 385′-ctaggtaccCTAGTCACTCCTCGT 50 39 5′-TGCGGTACCatggttgaaagagac 51 405′-CTAGGTACCttaggaactttgagc 52

Example 2-2: Confirmation of N-Acetylmethionine Production CapacityUsing Novel Acyltransferase

One colony of the transformed Escherichia coli prepared in Example 2-1was inoculated in 3 mL of an LB liquid medium containing 25 mg/L ofkanamycin and 0.2% (w/v) of glucose and cultured at 37° C. for 8 hours,and then inoculated in 50 mL of the same medium and cultured overnight,so as to obtain a culture solution. The culture solution was centrifugedto obtain pellets, the pellets were suspended in 5 mL of a 50 mMphosphate buffer (pH 7.0), and then cells were disrupted usingsonication to obtain cell debris. The cell debris was removed bycentrifugation at 14,000 rpm for 30 minutes to obtain a supernatant.Considering that the size of the novel acyltransferase is about 19 kDa,the filtrate was passed through an Amicon Ultra (Milipore, Ireland) 30kDa cut-off membrane and then through a 10 kDa cut-off membrane toobtain a concentrate remaining on the filter. The concentrate filled aHiTrap Q FF column (GE, USA) filled with Q sepharose, the novelacyltransferase was purely separated using NaCl concentration gradients(80, 100, 150, 200, 300, 500 mM, in that order). The diluted enzyme wasre-concentrated through an Am icon Ultra 10 kDa cut-off membrane. Thedegree of overexpression and purification of the novel acyltransferasewas confirmed by SDS-PAGE.

In order to analyze the activity of the purified novel acyltransferase,the amount of the N-acetylmethionine produced after introducing anenzyme concentrate into a pH 7.0 phosphate buffer containing 20 mMacetyl coenzyme A and 20 mM methionine and performing a reaction at 37°C. for 10 minutes was measured using HPLC (Shimadzu, system controllerCBM-20A and other accessories). Further, the concentration of thepurified novel acyltransferase was measured by a Bradford assay, andthen the produced N-acetylmethionine value was divided by the enzymeconcentration to compare the specific activity of the enzyme (Table 3).

Among the purified novel acyltransferases, the Pseudomonasputida-derived enzyme exhibits the highest activity of 3.8 U/mg, whichis 5 times specific activity (0.745 U/mg) of YncA disclosed in thedocument (U.S. Pat. No. 8,143,031 B2, Table 3). In addition, theBacillus subtilis-derived enzyme and the Enterobacter sp. 638-derivedenzyme exhibit activities of 1.6 U/mg and 1.7 U/mg, respectively, eachof which is 2 or more times specific activity of YncA. Each of thePseudovibrio sp. FO-BEG1-derived, Yarrowia lipolytica-derived, andCorynebacterium glutamicum-derived novel acyltransferases has lowspecific activity of less than I U/mg, but the expression amount thereofon SDS-PAGE is not so small. Therefore, the N-acetylmethionine producingcapacity of the transformant may be expected to be improved depending onthe degree of expression in the host (Table 3).

TABLE 3 Acyltransferase Specific activity (U/mg) Pseudomonasputida-derived 3.8 Bacillus subtilis-derived 1.6 Enterobacter sp.638-derived 1.7 Pseudovibrio sp. FO-BEG1-derived 0.4 Yarrowia lipolyticaPO1f-derived 0.4 Corynebacterium glutamicum-derived 0.8

Example 3: N-Acetylmethionine Conversion Reaction Using Pseudomonasputida-Derived Novel Acyltransferase Example 3-1: N-AcetylmethionineConversion Reaction Using Pseudomonas putida-Derived Purified NovelAcyltransferase

A methionine conversion reaction was performed using 1 mg of thePseudomonas putida-derived purified acyltransferase, which had thehighest activity in Example 2. 3 ml of a 50 mM phosphate buffer of pH7.0 containing 20 mM methionine and 20 mM acetyl coenzyme A was used asa substrate solution. When the reaction solution was analyzed by HPLC(SHIMADZU, SYSTEM CONTROLLER CBM-20A and other accessories) afterreaction for 3 hours under conditions of 37° C. and 150 rpm, theproduction of 3.1 g/L of N-acetylmethionine was confirmed. This is avalue obtained by converting methionine and acetyl coenzyme A in thereaction solution by 75% or more.

Example 3-2: N-Acetylmethionine Conversion Reaction of Cell ProvidedTherein with Pseudomonas putida-Derived Acyltransferase Depending onIncrease of Cell Membrane Permeability

One colony of the transformed Escherichia coli BL21(DE3)/pUCtk-ppmatmade by introducing the Pseudomonas putida-derived novel acyltransferasegene prepared in Example 2 was inoculated in 3 ml of an LB liquid mediumcontaining 25 mg/L of kanamycin and 1% (w/v) of glucose and cultured at37° C. for 8 hours to obtain a culture solution, and then 504 of theculture solution was inoculated in 50 mL of an LB liquid mediumcontaining 25 mg/L of kanamycin and 0.2% (w/v) of glucose and culturedovernight. The culture solution was centrifuged to obtain cell pellets,the cell pellets were frozen in a refrigerator at −20° C. The processesof remelting the fully frozen cell pellets at room temperature andrefreezing these cell pellets were repeated three times to impart highpermeability to the cell membrane. The culture solution was resuspendedto a total volume of 5 mL by adding a 50 mM phosphate buffer (pH 7.0) toconcentrate the culture solution. 1.8 mL of a 50 mM phosphate buffer (pH7.0) containing 2% (w/v) methionine; 1.8 mL of a 50 mM phosphate buffer(pH 7.0) containing 2% (w/v) methionine and 20 mM acetyl coenzyme A; and1.8 mL of a 50 mM phosphate buffer (pH 7.0) containing 2% (w/v)methionine and 2% (w/v) glucose were respectively put into 15 mL testtubes, and were each mixed with the enzyme bacteria having improved cellmembrane permeability obtained through the above procedures by 200 μL toa total volume of 2 mL, and were then reacted for 10 hours underconditions of 37° C. and 150 rpm. The purpose of adding glucose to thereaction solution is to increase the production amount of acetylcoenzyme A, which may be deficient in the reaction, and is not intendedfor the survival of Escherichia coli. The reaction solution was analyzedby HPLC (SHIMADZU, SYSTEM CONTROLLER CBM-20A and other accessories), andthe results thereof are given in Table 4. As given in Table 4, it wasfound that when the acetyl coenzyme A was added at the time offermentation, the molar conversion rate of the reaction solution wasincreased compared to when the acetyl coenzyme A was not added. Further,since it was found that the molar conversion rate of the reactionsolution was increased even when sucrose was added instead of the acetylcoenzyme A, it was estimated that the addition of glucose makes aportion of the acetyl coenzyme A. As the result of using strains eachhaving a high-permeability cell membrane, N-acetylmethionine was able tobe produced at high concentration, and was also able to be produced athigh concentration even when only glucose was added to the mediuminstead of the acetyl coenzyme A.

TABLE 4 Addition in reaction N-Acetylmethionine Molar conversion ratesolution concentration (g/L) (%) Not added 13.1 51 Acetyl coenzyme A20.0 78 Glucose 18.7 73

Example 4: Production of N-Acetyl-L-Methionine Through Fermentation ofTransformant Provided Therein with Novel Acyltransferase Example 4-1:Production of N-Acetyl-L-Methionine Through Escherichia coli ProvidedTherein with Novel Acyltransferase

One of the colonies of the six kinds of transformants prepared inExample 2 was inoculated in 3 mL of an LB liquid medium containing 25mg/L of kanamycin and 1% (w/v) of glucose and cultured at 37° C. for 8hours to obtain a culture solution, and then the culture solution wasinoculated in 50 mL of an LB liquid medium containing 25 mg/L ofkanamycin, 0.2% (w/v) of glucose, and 2% (w/v) of methionine andcultured overnight. In this case, as a control group, a pUCtk emptyvector was transformed into BL21 (DE3) and used. After the cells in theculture solution were removed by centrifugation, the producedN-acetylmethionine was analyzed using HPLC (SHIMADZU, SYSTEM CONTROLLERCBM-20A and other accessories). In the case of BL21(DE3)/pUCtk-ppmat,N-acetylmethionine was produced at the highest concentration of 3.03g/L. In the case of BL21(DE3)/pUCtk-entmat, N-acetylmethionine wasproduced at the second highest concentration of 2.23 g/L. Even in thecase of the empty vector, a trace amount of N-acetylmethionine wasdetected, which is presumed to be a role of the YncA enzyme possessed byEscherichia coli (Table 5).

TABLE 5 Concentration of N-acetylmethionine in Transformant culturesolution (g/L) BL21(DE3)/pUCtk <0.1 BL21(DE3)/pUCtk-ppmat 3.03BL21(DE3)/pUCtk-bsmat 1.60 BL21(DE3)/pUCtk-entmat 2.23BL21(DE3)/pUCtk-pvmat 0.17 BL21(DE3)/pUCtk-ylmat 0.13BL21(DE3)/pUCtk-cgmat 0.58

Example 4-2: Production of N-Acetyl-L-Methionine Through CorynebacteriumProvided Therein with Novel Acyltransferase Example 4-2-1: Preparationof Acyltransferase Overexpression Vector for Introducing Microorganismof Genus Corynebacterium

In order to examine the effect of N-acetyl-L-methionine production ofthe novel acyltransferases (SEQ ID NOS. 1, 2, 3, 4, 5, and 6) confirmedfrom Example 1 in microorganisms of genus Corynebacterium, vectors foroverexpressing the corresponding genes were prepared. A primer in whichan EcoRV restriction enzyme site is inserted at the 5′ end and a primerin which a PstI site is inserted at the 3′ end were synthesized based onbase sequences 7, 8, 9, 10, 11, and 12.

The gene based on base sequence 7 was polymerized through PCR using thevector pUCtk-ppmat of Example 1 as a template and using primers 5 and 6.The gene based on base sequence 8 was polymerized through PCR using thevector pUCtk-bsmat of Example 1 as a template and using primers 7 and 8.The gene based on base sequence 9 was polymerized through PCR using thevector pUCtk-entmat of Example 1 as a template and using primers 9 and10. The gene based on base sequence 10 was polymerized through PCR usingthe vector pUCtk-pvmat of Example 1 as a template and using primers 11and 12. The gene based on base sequence 11 was polymerized through PCRusing the vector pUCtk-ylmat of Example 1 as a template and usingprimers 13 and 14. The gene based on base sequence 12 was polymerizedthrough PCR using the vector pUCtk-cgmat of Example 1 as a template andusing primers 15 and 16.

As the promoter of the acyltransferase, a promoter cj7 (KoreanUnexamined Patent Application Publication No. 10-2004-0107215) was used.In order to obtain the DNA fragment of the cj7 promoter, a primer inwhich a KpnI restriction enzyme is inserted at the 5′ end and a primerin which an EcoRV site is inserted at the 3′ end were synthesized, andthe cj7 promoter was amplified through PCR using p117-cj1-gfp as atemplate (Korean Patent Application Publication No. 10-2004-0107215). Inthis case, PCR was performed by conducting denaturation at 94° C. for 5minutes, repeating denaturation at 94° C. for 30 seconds, annealing at56° C. for 30 seconds and polymerization at 72° C. for 1 minute 30times, and then conducting a polymerization reaction at 72° C. for 7minutes.

The six kinds of amplified acyltransferase polynucleotides were treatedwith restriction enzyme PstI to obtain DNA fragments, and the cj7promoter polynucleotide was treated with KpnI and EcoRV to obtain a DNAfragment. After six DNA fragments were obtained, they were linked to theKpn1 and Pst1 sites of the pECCG117 vector, which is a shuttle vector ofEscherichia and Corynebacterium, to be transformed into Escherichia coliDH5α, and were smeared in an LB solid medium containing kanamycin (25mg/L). The colonies transformed with the vector into which the gene wasinserted were selected by PCR, and then plasmids were obtained using agenerally known plasmid extraction method. The obtained plasmids werenamed pECCG117-Pcj7-ppmat, pECCG117-Pcj7-bsmat, pECCG117-Pcj7-entmat,pECCG117-Pcj7-pvmat, pECCG117-Pcj7-ylmat, and pECCG117-Pcj7-cgmat.

Example 4-2-2: Preparation of Strain for Introducing NovelAcyltransferase Overexpression Vector and Confirmation ofN-Acetyl-L-Methionine Production Capacity

Each of the vectors pECCG117-Pcj7-ppmat, pECCG117-Pcj7-bsmat,pECCG117-Pcj7-entmat, pECCG117-Pcj7-pvmat, pECCG117-Pcj7-ylmat, andpECCG117-Pcj7-cgmat, prepared in Example 4-2-1, and vector pECCG117 ofthe experimental control group was introduced into the Corynebacteriumglutamicum ATCC13032 using an electric pulse method, smeared in acomposite plate medium containing kanamycin (25 mg/L), and then culturedat 30° C. for 24 hours to obtain strains. The obtained strains werenamed 13032/pECCG117-Pcj7-ppmat, 13032/pECCG117-Pcj7-bsmat,13032/pECCG117-Pcj7-entmat, 13032/pECCG117-Pcj7-pvmat,13032/pECCG117-Pcj7-ylmat, 13032/pECCG117-Pcj7-cgmat, and13032/pECCG117. In order to confirm the N-acetylmethionine productioncapacity of the transformant, the strains were inoculated in 14 mL of adisposable culture tube including 3 mL of a composite liquid mediumcontaining kanamycin (25 mg/L), and shake-cultured for 24 hours underconditions of 30° C. and 200 rpm to obtain a seed culture solution. 1 mLof the seed culture solution was inoculated in a 250 mL corner-baffleflask including 24 mL of a composite liquid medium containing kanamycin(25 mg/L) and methionine (2% (20 g/L)), and shake-cultured for 24 hoursunder conditions of 37° C. and 200 rpm. After completing the culture,the concentration of N-acetylmethionine was analyzed using HPLC(SHIMADZU, SYSTEM CONTROLLER CBM-20A and other accessories) (Table 6).

In the case of the transformant 13032/pECCG117 prepared as a controlgroup, 1.07 g/L of N-acetylmethionine was produced. This result ispresumed to be caused by the ability of the acyltransferase possessed byCorynebacterium glutamicum, which is a parent strain. Further, it can beascertained that a larger amount of N-acetylmethionine is produced ascompared with original ability as the result of overexpressing the sixkinds of novel acyltransferases.

<Composite Plate Medium>

20 g glucose, 50 g (NH₄)₂SO₄, 10 g peptone, 5 g yeast extract, 1.5 gurea, 5 g KH₂PO₄, 10 g K₂HPO₄, 0.5 g MgSO₄7H₂O, 100 μg biotin, 1000 μgthiamine HCl, 2000 μg calcium pantothenate, 2000 μg nicotinamide, 20 gagar, and 25 mg kanamycin (based on 1 L distilled water)

<Composite Liquid Medium>

20 g glucose, 10 g peptone, 5 g yeast extract, 1.5 g urea, 4 g KH₂PO₄, 8g K₂HPO₄, 0.5 g MgSO₄7H₂O, 100 μg biotin, 1000 μg thiamine HCl, 2000 μgcalcium pantothenate, 2000 μg nicotinamide, and 25 mg kanamycin (basedon 1 L distilled water)

TABLE 6 Concentration of N-acetylmethionine in Transformant culturesolution (g/L) 13032/pECCG117 1.07 13032/pECCG117-Pcj7-ppmat 2.5013032/pECCG117-Pcj7-bsmat 1.79 13032/pECCG117-Pcj7-entmat 2.1113032/pECCG117-Pcj7-pvmat 1.23 13032/pECCG117-Pcj7-ylmat 1.3113032/pECCG117-Pcj7-cgmat 2.58

Example 4-3: Production of N-Acetyl-L-Methionine Through SaccharomycesProvided Therein with Novel Acyltransferase Example 4-3-1: Preparationof Acyltransferase Overexpression Vector for Saccharomyces

In order to examine the effect of N-acetyl-L-methionine production ofthe novel acyltransferases (SEQ ID NOS. 1, 2, 3, 4, 5, and 6) confirmedfrom Example 1 in Saccharomyces, vectors for overexpressing thecorresponding genes were prepared. A primer in which a SpeI restrictionenzyme site is inserted at the 5′ end and a primer in which a XhoI siteis inserted at the 3′ end were synthesized based on base sequences 7, 8,9, 10, 11, and 12.

The gene based on base sequence 7 was polymerized through PCR using thevector pUCtk-ppmat of Example 1 as a template and using primers 17 and18. The gene based on base sequence 8 was polymerized through PCR usingthe vector pUCtk-bsmat of Example 1 as a template and using primers 19and 20. The gene based on base sequence 9 was polymerized through PCRusing the vector pUCtk-entmat of Example 1 as a template and usingprimers 21 and 22. The gene based on base sequence 10 was polymerizedthrough PCR using the vector pUCtk-pvmat of Example 1 as a template andusing primers 23 and 24. The gene based on base sequence 11 waspolymerized through PCR using the vector pUCtk-ylmat of Example 1 as atemplate and using primers 25 and 26. The gene based on base sequence 12was polymerized through PCR using the vector pUCtk-cgmat of Example 1 asa template and using primers 27 and 28. In this case, the PCR wasperformed by conducting denaturation at 94° C. for 5 minutes, repeatingdenaturation at 94° C. for 30 seconds, annealing at 56° C. for 30seconds and polymerization at 72° C. for 1 minute 30 times, and thenconducting a polymerization reaction at 72° C. for 7 minutes.

The six kinds of amplified acyltransferase polynucleotides were treatedwith restriction enzymes SpeI and XhoI to obtain DNA fragments. Aftersix DNA fragments were obtained, they are linked to the SpeI and XhoIsites of the p414ADH vector, which is a shuttle vector of Escherichiaand Saccharomyces, to be transformed into Escherichia coli DH5a, andwere smeared in an LB solid medium containing ampicillin (100 mg/L). Thecolonies transformed with the vector into which the gene was insertedwere selected by PCR, and then plasmids were obtained using a plasmidminiprep kit (Bioneer, Korea). The obtained plasmids were namedp414ADH-ppmat, p414ADH-bsmat, p414ADH-entmat, p414ADH-pvmat,p414ADH-ylmat, and p414ADH-cgmat.

Example 4-3-2: Preparation of Strain for Introducing NovelAcyltransferase Overexpression Vector and Confirmation ofN-Acetyl-L-Methionine Production Capacity

Each of the vectors p414ADH-ppmat, p414ADH-bsmat, p414ADH-entmat,p414ADH-pvmat, p414ADH-ylmat, and p414ADH-cgmat, prepared in Example4-3-1, and vector pECCG117 of the experimental control group wasintroduced into Saccharomyces cerevisiae CEN.PK2-1D (Korean PatentRegistration No. 10-1577134), which is a typical wild yeast receivedfrom EUROSCARF, using a yeast transformation method. The vectors wereintroduced into the Saccharomyces cerevisiae CEN.PK2-1D, and thensmeared in a YPD plate medium (1% yeast extract, 2% bacto-peptone, 2%glucose), and cultured at 30° C. for 24 hours to obtain strains. Theobtained strains were named ScCEN/p414ADH-ppmat, ScCEN/p414ADH-bsmat,ScCEN/p414ADH-entmat, ScCEN/p414ADH-pvmat, ScCEN/p414ADH-ylmat,ScCEN/p414ADH-cgmat, and ScCEN/p414ADH. In order to confirm theN-acetylmethionine production capacity of the transformant, the strainswere inoculated in 14 mL of a disposable culture tube including 3 mL ofa YPD liquid medium containing ampicillin (100 mg/L), and shake-culturedfor 24 hours under conditions of 30° C. and 200 rpm to obtain a seedculture solution. 1 mL of the seed culture solution was inoculated in a250 mL corner-baffle flask including 24 mL of a YPD liquid mediumcontaining methionine (0.5% (5 g/L)), and shake-cultured for 24 hoursunder conditions of 30° C. and 200 rpm. After completing the culturing,the concentration of N-acetyl-L-methionine was analyzed using HPLC(SHIMADZU, SYSTEM CONTROLLER CBM-20A and other accessories) (Table 7).

In the case of the transformant 13032/pECCG117 prepared as a controlgroup, 1.07 g/L of N-acetylmethionine was produced. This result ispresumed to be caused by the ability of the acyltransferase possessed byCorynebacterium glutamicum, which is a parent strain. Further, it can beascertained that a larger amount of N-acetylmethionine is produced ascompared with the original ability as a result of overexpressing the sixkinds of novel acyltransferases.

As can be seen from the transformant ScCEN/p414ADH, it is presumed thatSaccharomyces cerevisiae itself does not produce N-acetylmethionine, butit was ascertained that the strains in which six kinds of novelacyltransferases are overexpressed have N-acetylmethionine productioncapacity.

TABLE 7 Concentration of N-acetylmethionine in Transformant culturesolution (g/L) ScCEN/p414ADH 0 ScCEN/p414ADH-ppmat 1.64ScCEN/p414ADH-bsmat 1.44 ScCEN/p414ADH-entmat 1.65 ScCEN/p414ADH-pvmat0.42 ScCEN/p414ADH-ylmat 1.40 ScCEN/p414ADH-cgmat 0.30

Example 4-4: Production of N-Acetyl-L-Methionine Through Yarrowiaprovided Therein with Novel Acyltransferase Example 4-4-1: Preparationof Acyltransferase Overexpression Vector for Yarrowia

In order to examine the effect of N-acetyl-L-methionine production ofthe novel acyltransferases (SEQ ID NOS. 1, 2, 3, 4, 5, and 6) confirmedfrom Example 1 in Yarrowia, vectors for overexpressing the correspondinggenes were prepared. A primer in which a KpnI restriction enzyme site isinserted at the 5′ end and a primer in which a KpnI restriction enzymesite is inserted at the 3′ end were synthesized based on base sequences7, 8, 9, 10, 11, and 12.

The gene based on base sequence 7 was polymerized through PCR using thevector pUCtk-ppmat of Example 1 as a template and using primers 29 and30. The gene based on base sequence 8 was polymerized through PCR usingthe vector pUCtk-bsmat of Example 1 as a template and using primers 31and 32. The gene based on base sequence 9 was polymerized through PCRusing the vector pUCtk-entmat of Example 1 as a template and usingprimers 33 and 34. The gene based on base sequence 10 was polymerizedthrough PCR using the vector pUCtk-pvmat of Example 1 as a template andusing primers 35 and 36. The gene based on base sequence 11 waspolymerized through PCR using the vector pUCtk-ylmat of Example 1 as atemplate and using primers 37 and 38. The gene based on base sequence 12was polymerized through PCR using the vector pUCtk-cgmat of Example 1 asa template and using primers 39 and 40. In this case, the PCR wasperformed by conducting denaturation at 94° C. for 5 minutes, repeatingdenaturation at 94° C. for 30 seconds, annealing at 56° C. for 30seconds and polymerization at 72° C. for 1 minute 30 times, and thenconducting a polymerization reaction at 72° C. for 7 minutes.

The six kinds of amplified acyltransferase polynucleotides were treatedwith restriction enzyme KpnI to obtain DNA fragments. After six DNAfragments were obtained, they are linked to the KpnI site of a shuttlevector pIMR53_AUX (FEMS Microbiology Letters Volume 199, Issue 1, pages97-102, May 2001) of Escherichia and Yarrowia, to be transformed intoEscherichia coli DH5α, and were smeared in an LB solid medium containingampicillin (100 mg/L). The colonies transformed with the vector intowhich the gene was inserted were selected by PCR, and then plasmids wereobtained using a plasmid miniprep kit (Bioneer, Korea). The obtainedplasmids were named pIMR53U-ppmat, pIMR53U-bsmat, pIMR53U-entmat,pIMR53U-pvmat, pIMR53U-ylmat, and pIMR53U-cgmat.

Example 4-4-2: Preparation of Strain for Introducing AcyltransferaseOverexpression Vector and Confirmation of N-Acetyl-L-MethionineProduction Capacity

Each of the vectors pIMR53U-ppmat, pIMR53U-bsmat, pIMR53U-entmat,pIMR53U-pvmat, pIMR53U-ylmat, and pIMR53U-cgmat, and vector pIMR53_AUXof the experimental control group was introduced into Yarrowialipolytica PO1f (ATCC MYA-2613™) purchased from American type CultureCollection using a yeast transformation method. The vectors wereintroduced into the Yarrowia lipolytica PO1f, and then smeared in a YPDplate medium (1% yeast extract, 2% bacto-peptone, 2% glucose), andcultured at 30° C. for 24 hours to obtain strains. The obtained strainswere named Yl/pIMR53U-ppmat, Yl/pIMR53U-bsmat, Yl/pIMR53U-entmat,Yl/pIMR53U-pvmat, Yl/pIMR53U-ylmat, Yl/pIMR53U-cgmat, and Yl/pIMR53U. Inorder to confirm the N-acetylmethionine production capacity of thetransformant, the strains were inoculated in 14 mL of a disposableculture tube including 3 mL of a YPD liquid medium, and shake-culturedfor 24 hours under conditions of 30° C. and 200 rpm to obtain a seedculture solution. I mL of the seed culture solution was inoculated in a250 mL corner-baffle flask including 24 mL of a YPDm liquid medium (10 gglucose, 3.28 g Na₂HPO₄, 3.22 g NaH₂PO₄, 2 g yeast extract, and 50 g/LProteose-peptone) containing methionine (0.5% (5 g/L)), andshake-cultured for 24 hours under conditions of 30° C. and 200 rpm.After completing the culturing, the concentration ofN-acetyl-L-methionine was analyzed using HPLC (SHIMADZU, SYSTEMCONTROLLER CBM-20A and other accessories) (Table 8).

It is presumed that N-acetylmethionine is also produced in thetransformant Yl/pIMR53U due to the effect of the acyltransferasepossessed by the wild type of Yarrowia lipolytica, but it wasascertained that the strains in which six kinds of novelacyltransferases are overexpressed have N-acetylmethionine productioncapacity.

TABLE 8 Concentration of N-acetyl-L-methionine Transformant in culturesolution (g/L) YI/pIMR53U 1.02 YI/pIMR53U-ppmat 1.92 YI/pIMR53U-bsmat1.78 YI/pIMR53U-entmat 1.82 YI/pIMR53U-pvmat 1.26 YI/pIMR53U-ylmat 2.01YI/pIMR53U-cgmat 1.38

From the above results, it is suggested that acyltransferases newlydeveloped in the present disclosure and microorganisms including thesame efficiently produce N-acetyl-L-methionine as compared with a knownacyltransferase YncA.

As described above, those skilled in the art will be able to understandthat the present disclosure can be easily executed in other detailedforms without changing the technical spirit or an essential featurethereof. Therefore, it should be appreciated that the aforementionedembodiments are illustrative in all aspects and are not restricted. Thescope of the present disclosure is represented by claims to be describedbelow rather than the detailed description, and it is to be interpretedthat the meaning and scope of the claims and all changes or modifiedforms derived from the equivalents thereof come within the scope of thepresent disclosure.

1. A microorganism having an acyltransferase activity, the microorganismcomprising a polypeptide represented by an amino acid sequence of anyone of SEQ ID NOS. 1 to 6 or an amino acid sequence having 90% or morehomology to the amino acid sequence.
 2. The microorganism according toclaim 1, wherein the microorganism is selected from the group consistingof Escherichia sp., Corynebacterium sp., Saccharomyces sp., and Yarrowiasp.
 3. The microorganism according to claim 1, wherein the microorganismhas an acetyltransferase activity to L-methionine and thus producesN-acetyl-L-methionine.
 4. A polypeptide having an acetyltransferaseactivity, the polypeptide being represented by an amino acid sequence ofany one of SEQ ID NOS. 1 to 6 or an amino acid sequence having 90% ormore homology to the amino acid sequence.
 5. A polynucleotide encodingthe polypeptide of claim
 4. 6. An expression vector comprising thepolynucleotide of claim
 5. 7. A composition for preparingN-acetyl-L-methionine from L-methionine, the composition comprising, asan active ingredient: (i) the polypeptide of claim 4; (ii) amicroorganism comprising the polypeptide or a culture of themicroorganism; or a combination thereof.
 8. A method of preparingN-acetyl-L-methionine, comprising: acetylating L-methionine using (i)the polypeptide of claim 4; (ii) a microorganism comprising thepolypeptide or a culture of the microorganism; or a combination thereof.9. The method according to claim 8, further comprising: recoveringN-acetyl-L-methionine, which is the acetylated L-methionine.
 10. Thecomposition of claim 7, wherein the microorganism is selected from thegroup consisting of Escherichia sp., Corynebacterium sp., Saccharomycessp., and Yarrowia sp.
 11. The composition of claim 7, wherein themicroorganism has an acetyltransferase activity to L-methionine and thusproduces N-acetyl-L-methionine.
 12. The method of claim 8, wherein themicroorganism is selected from the group consisting of Escherichia sp.,Corynebacterium sp., Saccharomyces sp., and Yarrowia sp.
 13. The methodof claim 8, wherein the microorganism has an acetyltransferase activityto L-methionine and thus produces N-acetyl-L-methionine.
 14. The methodof claim 12, further comprising: recovering N-acetyl-L-methionine, whichis the acetylated L-methionine.
 15. The method of claim 13, furthercomprising: recovering N-acetyl-L-methionine, which is the acetylatedL-methionine.